A striking number of hereditary nervous system diseases are caused by deleterious mutations in poorly characterized enzymes from the serine hydrolase class. Over the past decade, our group has developed an innovative set of chemical proteomic and metabolomic platforms for assigning functions to uncharacterized serine hydrolases and have a special interest in applying these platforms to serine hydrolases that are causally linked by human genetics to nervous system diseases. In this grant application, we focus on the role that serine hydrolases play in the neurological disorder PHARC (polyneuropathy, hearing loss, ataxia, retinosis pigmentosa, and cataract). Specifically, we have determined that the serine hydrolase ABHD12, mutations of which cause PHARC, is a major brain lyso-phosphatidylserine (lyso-PS) lipase. ABHD12-/- mice exhibit elevated brain lyso-PS content and auditory and motor deficits coupled with heightened neuroinflammation, implicating deregulated lyso-PS signaling as a contributory factor to PHARC. Blocking the upstream enzyme(s) that produces lyso-PS could thus provide a therapeutic strategy to treat PHARC and possibly other (neuro) inflammatory diseases. We recently determined that the heretofore uncharacterized serine hydrolase ABHD16A is a major PS lipase responsible for generating lyso-PS in mammalian systems, including mouse brain and PHARC subject-derived lymphoblasts. The goal of this application is to perform a high-throughput screen of TSRI's 640,000+ compound library to identify structurally novel classes of ABHD16A inhibitors that can be progressed to selective and in vivo-active chemical probes. We have established a diverse and innovative set of biochemical, chemoproteomic, and lipidomic assays to rigorously assess the potency, selectivity, and cellular activity of ABHD16A inhibitors. We expect that the Specific Aims of this application will deliver multiple structurally distinct ABHD16A inhibitors that show suitable levels of potency (in vitro IC50 < 1 M), selectivity (a limited number of off-targets (lss than five) across the serine hydrolase class), and cellular activity (in situ IC50 < 10 M) for prioritization as leads for medicinal chemistry optimization into in vivo-active chemical probes. Our collaborative research team has a strong track-record of performing high-throughput screens on serine hydrolases and developing leads from these screens into selective and in vivo-active inhibitors. We are therefore well-equipped to progress the prioritized inhibitors discovered in this application toward optimized chemical probes for testing the function of ABHD16A and its lyso-PS products in PHARC and other (neuro) inflammatory disorders.